A comparison between NMR and GCMS 13C-isotopomer analysis in cardiac metabolism

Abstract Anhydrous enol-oxaloacetate (AEO) has demonstrated the ability to enhance neuronal cell bioenergetics and activate brain mitochondrial biogenesis. Since oxaloacetate has demonstrated positive effects on brain bioenergetics in neurodegenerative diseases we have begun to investigate whether AEO may also have a positive effect on the altered cellular metabolism found in cancer cells, particularly Glioblastoma multiforme. The “Warburg effect” describes an abnormal metabolic state in cancer, distinct from normal tissue, in which energy is generated through enhanced conversion of pyruvate to lactate even in the presence of oxygen during glycolysis. Oxaloacetate (OAA) is a key anaplerotic substrate that is required to maintain TCA cycle flux. The role of oxaloacetate supplementation on the energy metabolism is not known in cancer cells. Goal of this study is to investigate the changes in metabolic fluxes in glucose metabolism with and without the presence of OAA in patient-derived GBM cells. We use GC-MS based 13C isotopomer analysis for this study. GBM cells are grown in 15mM glucose containing DMEM medium supplemented with 2mM oxaloacetate for 10 days. 6 hours prior to harvesting, [U-13C]glucose is introduced to the medium. 13C isotopomer analysis of GC-MS data showed that OAA supplementation for 10 days drastically decreased Warburg glycolysis by reducing 13C labeling (M+3) by 19.7% and 48.8% in pyruvate and lactate pools respectively in comparison with cells not treated with OAA. M+3 13C labeled pyruvate entered TCA cycle via acetyl-CoA, where we also observed reduced levels of M+2 13C labeled citrate (20.5%) and glutamate (23.9%) isotopomers. Pyruvate can also enter TCA cycle via pyruvate carboxylation pathway and this activity was also found to be slightly decreased in the OAA treated cells. All the differences were statistically significant. These results indicate that OAA can be used to alter bioenergetics of GBM cells, specifically glucose oxidation.

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Pattern of substrate utilization in vascular smooth muscle using 13C isotopomer analysis of glutamate

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1998.275.6.h2227 ◽

1998 ◽

Vol 275 (6) ◽

pp. H2227-H2235 ◽

Cited By ~ 4

Author(s):

Tara M. Allen ◽

Christopher D. Hardin

Keyword(s):

Smooth Muscle ◽

Vascular Smooth Muscle ◽

Tca Cycle ◽

Tricarboxylic Acid ◽

Acetyl Coa ◽

Contractile State ◽

The Tca Cycle ◽

Direct Measurements ◽

Isotopomer Analysis ◽

13C Isotopomer Analysis

Although vascular smooth muscle (VSM) derives the majority of its energy from oxidative phosphorylation, controversy exists concerning which substrates are utilized by the tricarboxylic acid (TCA) cycle. We used 13C isotopomer analysis of glutamate to directly measure the entry of exogenous [13C]glucose and acetate and unlabeled endogenous sources into the TCA cycle via acetyl-CoA. Hog carotid artery segments denuded of endothelium were superfused with 5 mM [1-13C]glucose and 0–5 mM [1,2-13C]acetate at 37°C for 3–12 h. We found that both resting and contracting VSM preferentially utilize [1,2-13C]acetate compared with [1-13C]glucose and unlabeled substrates. The entry of glucose into the TCA cycle (30–60% of total entry via acetyl-CoA) exhibited little change despite alterations in contractile state or acetate concentrations ranging from 0 to 5 mM. We conclude that glucose and nonglucose substrates are important oxidative substrates for resting and contracting VSM. These are the first direct measurements of relative substrate entry into the TCA cycle of VSM during activation and may provide a useful method to measure alterations in VSM metabolism under physiological and pathophysiological conditions.

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Triidothyronine and epinephrine rapidly modify myocardial substrate selection: a 13C isotopomer analysis

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.2001.281.5.e983 ◽

2001 ◽

Vol 281 (5) ◽

pp. E983-E990 ◽

Cited By ~ 26

Author(s):

Julia J. Krueger ◽

Xue-Han Ning ◽

Barisa M. Argo ◽

Outi Hyyti ◽

Michael A. Portman

Keyword(s):

Direct Action ◽

Tricarboxylic Acid Cycle ◽

Substrate Selection ◽

Acetoacetic Acid ◽

Rat Hearts ◽

Isotopomer Analysis ◽

Myocardial Substrate ◽

13C Isotopomer Analysis ◽

High Work ◽

C Nmr

Triiodothyronine (T3) exerts direct action on myocardial oxygen consumption (MV˙o 2), although its immediate effects on substrate metabolism have not been elucidated. The hypothesis, that T3 regulates substrate selection and flux, was tested in isovolumic rat hearts under four conditions: control, T3 (10 nM), epinephrine (Epi), and T3 and Epi (TE). Hearts were perfused with [1,3-13C]acetoacetic acid (AA, 0.17 mM),l-[3-13C]lactic acid (LAC, 1.2 mM), U-13C-labeled long-chain free fatty acids (FFA, 0.35 mM), and unlabeled d-glucose (5.5 mM) for 30 min. Fractional acetyl-CoA contribution to the tricarboxylic acid cycle (Fc) per substrate was determined using 13C NMR and isotopomer analysis. Oxidative fluxes were calculated using Fc, the respiratory quotient, and MV˙o 2. T3increased ( P < 0.05) FcFFA, decreased FcLAC, and increased absolute FFA oxidation from 0.58 ± 0.03 to 0.68 ± 0.03 μmol · min−1 · g dry wt−1( P < 0.05). Epi decreased FcFFA and FcAA, although FFA flux increased from 0.58 ± 0.03 to 0.75 ± 0.09 μmol · min−1 · g dry wt−1. T3 moderated the change in FcFFA induced by Epi. In summary, T3 exerts direct action on substrate pathways and enhances FFA selection and oxidation, although the Epi effect dominates at a high work state.

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